Environmental hazards and stress, carcinogens, and anti-cancer therapeutics cause apoptotic cell death through the intrinsic or mitochondrial death pathway. Abnormal apoptotic response often contributes to tumor development and defects in apoptosis are intimately associated with tumor cell resistance to anti- neoplastic agents. Caspase activation lies in the core of apoptotic cell death. In the mitochondrial death pathway, cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in caspase-9 (an initiator caspase) activation, which ultimately activates effector caspases such as caspase-3 to execute cell demise. How Apaf-1 apoptosome is formed and regulated in vivo (i.e., in the stimulated cells) remains poorly understood. Recently, we provided evidence that physiological levels of nucleotides inhibit the CC-induced, apoptosome-mediated caspase-9 activation by binding directly to CC and preventing CC from interacting with Apaf-1 (Chandra et al., Cell 125, 1333-1346, 2006;Appendix I). Consequently, the CC- mediated apoptosome assembly and activation are blocked. Co-microinjection of nucleotides and CC renders cells resistant to the CC-induced apoptosis in vivo whereas experimentally reducing nucleotides enhances both CC and apoptotic stimuli-induced cell death. These observations lead us to hypothesize that physiological levels of nucleotides, in addition to their well-established roles in nucleic acid synthesis, intermediate metabolism, and maintenance of bioenergetics, also function as critical prosurvival factors by directly inhibiting the CC-mediated apoptosome formation and caspase activation. There are several critical unanswered questions related to Apaf-1 apoptosome regulation by nucleotides. We propose the following three Specific Aims to address some of these questions: 1) To further study nucleotide interaction with CC in vitro and to elucidate nucleotide interaction with CC in vivo;2) To investigate nucleotide interaction with Apaf-1 and its impact on apoptosome activation;and 3) To study the effects of Apaf-1 isoform expression and nucleotide interaction on Apaf-1 apoptosome activation. These goals will be achieved by a combination of cell biological, biochemical, and molecular approaches. The accomplishment of the proposed goals will greatly advance our understanding of apoptosome regulation in vivo and help us understand cellular response to environmental stress and cancer cell response to anti- tumor therapeutics.